Tn6198, a novel transposon containing the trimethoprim resistance gene dfrG embedded into a Tn916 element in Listeria monocytogenes

Tn6198, a novel transposon containing the trimethoprim resistance

gene dfrG embedded into a Tn916 element in Listeria monocytogenes

David Bertsch

_{, Anaı¨s Uruty}

_{, Janine Anderegg}

_{, Christophe Lacroix}

_{, Vincent Perreten}

_{and Leo Meile}

_*

1

_{Laboratory of Food Biotechnology, Institute of Food, Nutrition and Health, ETH Zurich, Schmelzbergstrasse 7, 8092 Zurich, Switzerland;}

Institute of Veterinary Bacteriology, University of Bern, La¨nggass-Strasse 122, 3012 Bern, Switzerland

*Corresponding author. Tel:+41-446323362; Fax: +41-446321403; E-mail: leo.meile@hest.ethz.ch

Received 22 October 2012; returned 9 November 2012; revised 12 December 2012; accepted 16 December 2012

Objectives: To characterize Tn6198, a novel conjugative transposon from the clinical Listeria monocytogenes

strain TTH-2007, which contains the tetracycline and trimethoprim resistance genes tet(M) and dfrG,

respect-ively, and to assess its transferability in vitro and in situ.

Methods: The complete sequence of Tn6198 was determined using a primer walking strategy. Horizontal gene

transfer studies were performed by filter matings, as well as on the surface of smear-ripened cheese and

smoked salmon. The presence of Tn916-like circular intermediates was determined by PCR. Antibiotic resistance

was determined by the broth microdilution method and microarray hybridization.

Results: Sequencing of Tn6198 revealed that a 3.3 kb fragment containing dfrG was integrated between

open reading frames 23 and 24 of Tn916. Furthermore, an additional copy of Tn916 was present in

L. monocytogenes TTH-2007. Both elements were transferred simultaneously and separately in vitro to

recipi-ents L. monocytogenes 10403S and Enterococcus faecalis JH2-2 by conjugation, resulting in either

tetracycline-and trimethoprim-resistant or solely tetracycline-resistant transconjugants. On the surface of cheese tetracycline-and

salmon, only L. monocytogenes 10403S transconjugants were detected.

Conclusions: This study reports the first Tn916-like element associated with a trimethoprim resistance gene, as

well as the first fully characterized transposon conferring multidrug resistance in L. monocytogenes. This is of

concern, as trimethoprim is administered to listeriosis patients with b-lactam allergy and as Tn6198 has a large

potential for dissemination, indicated by both intra-species and inter-genus transfer.

Keywords: tet(M), L. monocytogenes, conjugative transposon, multidrug resistance, horizontal gene transfer

Introduction

The Tn916 family of conjugative transposons is a growing group

of mobile genetic elements and is widespread mainly among

Fir-micutes.

Tn916, originally discovered in an Enterococcus faecalis

strain, was transferable in the absence of plasmids.

Most

members harbour the tetracycline resistance gene tet(M) which

encodes a ribosomal protection protein. However, Tn6000 from

Enterococcus casseliflavus

_{contains the tet(S) gene and is}

wide-spread among enterococci,

whereas the previously assumed

tet(S) gene in Tn916S from Streptococcus intermedius

was

recently shown to be a mosaic tet(S/M) gene.

Interestingly,

next to the many more Tn916-like elements with additional

re-sistance genes that have been reported,

_{no resistance genes}

could be identified in Tn5386 from Enterococcus faecium.

Tetracycline resistance, whenever detected in Listeria

mono-cytogenes isolates, is mostly associated with the tet(M) gene

located on Tn916

and less frequently with tet(S) carried by

plasmids.

Transferability of the tet(M) gene from L.

monocyto-genes was first shown for foodborne isolates in Italy.

_Generally,

the prevalence of L. monocytogenes with acquired resistance is

relatively low.

However, these strains can hamper the difficult

treatment of human listeriosis, a disease that seems to be

in-creasing, particularly in elderly people.

_{It was estimated that}

in 99% of the cases people had been infected with L.

monocyto-genes after ingestion of contaminated foodstuffs.

_Especially

when these bacteria are present on ready-to-eat foods like

smoked salmon or cheese, the lack of a heating step before

con-sumption constitutes a potential health risk. Therefore, next to

the human gut, where resistance gene transfer to Listeria spp.

most probably can occur, as demonstrated in a gut model,

food matrices also have to be taken into account as platforms

for horizontal gene transfer due to high bacterial concentrations.

Conjugative transfer of erythromycin and tetracycline resistance

#_{The Author 2013. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.} For Permissions, please e-mail: journals.permissions@oup.com

J Antimicrob Chemother 2013; 68: 986 – 991

genes from E. faecalis to enterococci, pediococci and

coagulase-negative staphylococci during sausage fermentation was

recent-ly demonstrated.

L. monocytogenes is naturally susceptible to trimethoprim;

therefore, this bacteriostatic antibiotic is often administered

together with sulfamethoxazole as co-trimoxazole to patients

with allergies to penicillins.

_{Resistance of L. monocytogenes}

against trimethoprim was first detected in France in an

envi-ronmental isolate containing the dfrD gene encoded on

the 3.7 kb plasmid pIP823.

Recently, two further isolates

from France were reported to contain this resistance gene,

namely a human clinical isolate

and a foodborne isolate.

The foodborne isolate additionally harboured the tet(M) gene

on a Tn916-like transposon. So far, there are no reports of

resist-ance in Listeria spp. caused by dfrG, a gene first detected in

Staphylococcus aureus

and highly prevalent in Staphylococcus

pseudointermedius.

The aim of this study was to characterize the molecular

mechanism of the trimethoprim and tetracycline resistance in

a clinical isolate of L. monocytogenes and to determine

whether both resistances were transferable by conjugation.

Materials and methods

Bacterial strains

Strain TTH-2007 was isolated by haemoculture in 2007 in Switzerland and provided by Professor Jacques Bille from the Centre Hospitalier Uni-versitaire Vaudois (CHUV) in Lausanne. Strains used in this study were cul-tured on brain heart infusion (BHI) agar medium (Biolife Italiana S.r.l., Milan, Italy) at 378C and stored in BHI medium containing 33% glycerol at 2808C.

DNA extraction and PCR assays

Genomic bacterial DNA was isolated as described before.23 _The

GeneJetTM _{Plasmid Miniprep Kit (Fermentas) with lysozyme (10 g/L as}

final concentration) treatment at 378C for 30 min was used to isolate cir-cular intermediates of transposons from 2 mL of overnight cultures grown in BHI with 26 mg/L tetracycline, as well as to investigate the pres-ence of plasmids.

Primers were purchased from Microsynth AG (Balgach, Switzerland) and Sanger sequencing analyses were performed with the cycle sequen-cing method by Microsynth AG and GATC Biotech AG (Konstanz, Germany) using capillary electrophoresis. DNA was previously purified with the GFXTM _{PCR DNA and Gel Band Purification Kit (GE Healthcare). PCR}

assays with primers used in this study are listed in Table S1 (available as Supplementary data at JAC Online). PCR Master Mix (2×) with Taq DNA polymerase (Fermentas) was applied for templates up to 3 kb; for longer targets GoTaqw Long PCR Master Mix (Promega) or PhusionTM

High-Fidelity DNA Polymerase (New England BioLabs) was used.

Phenotypic and genotypic characterization of

antibiotic resistance

Antibiotic resistance phenotypes were investigated by broth microdilution susceptibility testing in cation-adjusted Mueller–Hinton broth (Becton, Dickinson and Company) with 2.5% (volume/volume) laked horse blood (Oxoid) at 378C for 48 h, based on the CLSI guidelines M45-A2 for L. monocytogenes.24_{The following antibiotics were tested: minocycline,}

rifampicin, streptomycin, tetracycline and trimethoprim. L. monocyto-genes DSM 20600T_{and Streptococcus pneumoniae ATCC 46919 were}

used as reference/quality control strains. Antibiotic resistance genotypes were determined with biotin-labelled DNA hybridizing on a custom-made microarray (AMR+ve-2 ArrayTubesTM_{, Alere Technologies GmbH, Jena,}

Germany) targeting . 100 antibiotic resistance genes of Gram-positive bacteria, including dfrG. This microarray was based on a previous publica-tion.25 _{PCR assays (Table S1, available as Supplementary data at JAC}

Online) were applied to confirm the presence of antibiotic resistance genes, as well as of the transposon integrase gene int.

Antibiotic resistance gene transfer by filter mating

In vitro conjugation experiments on nitrocellulose membrane filters (0.45 mm; Millipore AG) were performed as described before26 _using

streptomycin-resistant L. monocytogenes 10403S27 _and

rifampicin-resistant E. faecalis JH2-228_{as recipients. Briefly, 1% of overnight cultures}

of donor and recipient were incubated in fresh BHI medium for 6 h before conjugation experiments, a mixture (1 :3) was passed through a sterile filter followed by a washing step with 2 mL of diluent [0.85% NaCl, 0.1% peptone from casein (VWR), pH 8.0] and incubation overnight at 378C. Cells were removed from the filter with 2 mL of diluent in a 50 mL reaction tube under shaking on a vortex-mixer. Isolation of trans-conjugant and donor colonies was obtained by spreading appropriate dilutions onto selective agar medium, supplemented with corresponding antibiotics (13 mg/L rifampicin, 26 mg/L streptomycin, 26 mg/L tetracyc-line and 13 mg/L trimethoprim). L. monocytogenes transconjugants were selected on PALCAM agar (Oxoid) at 378C and E. faecalis transconjugants were selected on DifcoTM_{KF Streptococcus Agar (Becton, Dickinson and}

Company) at 438C, respectively. The transfer rate was calculated as the number of transconjugants per donor.

Antibiotic resistance gene transfer on salmon and

cheese surfaces

In situ conjugation experiments were performed on the surface of smoked salmon and of smear-ripened cheese on the basis of the filter mating experiments. Swiss semi-hard cheese with a fully developed smear after a medium ripening time (90 days) was used. Cells of donor and recipients were grown for 6 h in BHI medium, 2 mL were centrifuged and the pellets were resuspended in the same volume of diluent. There-after, 0.1 mL of donor and 0.3 mL of recipient suspensions (106_–

107_{cells each) were spread onto the salmon or cheese surface (about}

70 cm2_{), respectively, and the food samples were stored for 5 days in}

closed boxes containing distilled water in a side compartment to retain a humid environment. Salmon samples were stored both at 98C and 228C, respectively, and cheese samples at 228C. The concentration of inocula was determined by plating onto BHI agar medium and calculated as cfu/cm2_{. After the incubation period, the cheese smear was scraped}

off with a sterile knife and stomached in 10 mL of diluent, whereas the complete salmon sample (15–20 g) was stomached in 20 mL of diluent. Appropriate dilutions were plated onto selective agar medium.

Identification of transconjugants

After every mating experiment, three donor colonies and up to 10 trans-conjugant colonies were isolated from corresponding selective agar media. PCR assays (Table S1, available as Supplementary data at JAC Online) were applied to determine species affiliation, as well as presence of antibiotic resistance genes. Phenotypic resistance was tested by the broth microdilution method as described above. As both TTH-2007 and 10403S are L. monocytogenes strains with serovar 1/2a, transconjugants were differentiated from donor and recipient strains by the presence of antibiotic resistance genes and rep-PCR fingerprinting, amplifying repeti-tive extragenic palindromic elements, which allows discrimination between bacterial strains.29 _{Furthermore, the highly polymorphic}

virulence gene actA was partially sequenced, as it allows effective differ-entiation of L. monocytogenes strains.30_{Presence of circular forms of}

Tn916-like transposons was tested by PCR (Table S1, available as Supple-mentary data at JAC Online).

Results

Characterization and sequencing of Tn6198

L. monocytogenes TTH-2007 exhibited phenotypic resistances to

tetracycline (64 mg/L), minocycline (32 mg/L) and trimethoprim

(.320 mg/L), which were attributed to tet(M) and dfrG,

respect-ively. Presence of the transposon integrase gene int and absence

of plasmids suggested association with the Tn1545-Tn916 family

of conjugative transposons.

By long PCR with combinations of

primers derived from tet(M), dfrG, int and Tn916 (GenBank

acces-sion no. U09422) together with a primer walking strategy, most

of the sequence of the novel transposon was obtained. Ring

closure was achieved with DNA of its circular intermediate.

Tn6198 had a length of 21322 bp (GenBank accession no.

JX120102) and comparison with Tn916 (Figure

1

a) revealed the

integration of a 3285 bp DNA fragment including the dfrG

gene between open reading frames (ORFs) 23 and 24 of Tn916.

This fragment (named F23-24) was identical to a nucleotide

sequence (GenBank accession no. AB205645) of S. aureus

strain CM.S2, in which the trimethoprim resistance dihydrofolate

reductase gene dfrG was first described.

_{Therefore, the ORFs}

next to dfrG were named orfU1 as designated in the

above-mentioned publication and orfU2, respectively (Figure

1

a).

Apart from dfrG fragment F23-24, the sequences of Tn6198

and Tn916 share a similarity of .99.9%.

Direct repeat sequences of 11 bp, which mark the point of

in-sertion of dfrG fragment F23-24, as well as inverted repeats of

27 bp were located (Figure

1

b). The circular form of this fragment

was detected by PCR with primers A and B (Figure

1

a and

Figure S1, available as Supplementary data at JAC Online).

Furthermore, by using an established PCR assay including

primers L1 and R1

we demonstrated the presence of the

circu-lar form of a Tn916-like antibiotic resistance carrier in TTH-2007

(Figure S1). However, the location of primer R1 to the left of the

inserted dfrG fragment F23-24 (Figure

1

a) did not enable

differ-entiation between the circular intermediate of Tn916 and

Tn6198. Therefore, a PCR assay with primers C and D was

applied leading to both a DNA fragment of 770 bp lacking the

dfrG insertion (Figure

1

c and Figure S1, available as

Supplemen-tary data at JAC Online) and a fragment of about 4 kb containing

the insertion (Figure

1

d and Figure S1). This demonstrated the

presence of an additional copy of Tn916 next to Tn6198 in

strain TTH-2007. The detection of two different joint sequences,

which are known to be formed by the flanking regions of a

trans-poson before its excision,

confirmed the presence of both

transposons. The 6 bp sequence between the termini of Tn916

comprised the bases AAGTAA and for Tn6198 it comprised the

bases ATTATA (nucleotides 1 –6 in JX120102).

In vitro and in situ gene transfer studies

MICs of antibiotics obtained by broth microdilution testing were a

prerequisite for correct experimental selection of donor and

recipient cells. Strain TTH-2007 showed high MIC values for

tetra-cycline and trimethoprim (see above), but low levels for

rifam-picin (0.016 mg/L) and streptomycin (0.25 mg/L). Recipient

L. monocytogenes 10403S had a high MIC value of streptomycin

(.256 mg/L) and E. faecalis JH2-2 of rifampicin (32 mg/L),

re-spectively; however, both were susceptible to tetracycline and

trimethoprim with values

≤0.5 mg/L.

Horizontal gene transfer was observed between L.

monocyto-genes strains TTH-2007 and 10403S after mating on filters,

where transfer rates between 1.3×10

_{and 3.0×10}

transcon-jugants per donor were detected. We were able to assign

trans-conjugant colonies to strain 10403S because of an additional

(a)

(b) (c) (d)

Figure 1. (a) Comparative schematic genetic organization of E. faecalis transposon Tn916 (GenBank accession no. U09422) and the novel L. monocytogenes transposon Tn6198 (GenBank accession no. JX120102). Grey areas indicate regions with .99.9% sequence identity. Black arrows indicate ORFs in Tn916 nomenclature. Grey arrows show ORFs of the integrated dfrG fragment F23–24 and black vertical lines indicate its insertion site, flanked by direct repeats (DR) and inverted repeats (IRL and IRR). Lengths of transposons are illustrated in kb and primers (A, B, C, D, L1 and R1) by small black arrows. (b) Sequences of direct and inverted repeats (DR, IRL and IRR); the black vertical line indicates the integration site. (c) PCR product with primers C and D for Tn916, without dfrG fragment F23-24. (d) PCR product with primers C and D for Tn6198, with dfrG fragment F23-24.

band at about 350 bp in the fingerprint obtained by rep-PCR

compared with strain TTH-2007. Furthermore, 2% difference in

their actA sequences enabled distinction between both strains

(data not shown). In the in situ experiments on salmon and

cheese surfaces, the concentration of the donor and recipient

strains was stable during the incubation time (10

–10

cfu/

cm

_{) and transconjugants were obtained with transfer rates of}

10

_{– 10}

_{transconjugants per donor, similar to the filter}

mating experiments. There was no significant difference

between storage of the salmon at 98C or 228C. Fewer

transcon-jugants were obtained when E. faecalis JH2-2 was used as

recipi-ent strain in the filter matings, resulting in a transfer rate of 10

.

No E. faecalis transconjugants were found on the salmon or

cheese surface and enterococcal counts decreased up to 2 logs

during storage.

Both Tn6198 and Tn916 were present in the donor, so we

tested whether the transposons were transferable together or

separately in vitro and in situ. Therefore, selection of

transconju-gants was performed by application of 26 mg/L tetracycline

and 13 mg/L trimethoprim, as well as 26 mg/L tetracycline or

13 mg/L trimethoprim alone, respectively. The different types

of transconjugants are listed in Table

1

. Interestingly, the two

transposons were detected together in several transconjugants

selected with tetracycline and trimethoprim, although Tn6198

alone already confers resistance to both antibiotics. In contrast,

after selection with only 13 mg/L trimethoprim, all

transconju-gants harboured Tn6198 but no additional copy of Tn916.

Selec-tion with 26 mg/L tetracycline alone led to three different types

of transconjugants, including transfer of solely Tn916, providing

only resistance to tetracycline.

The sequence of Tn6198 obtained from the donor strain

TTH-2007 (Figure

1

a) was identical to the corresponding

sequence in an L. monocytogenes transconjugant to which

Tn916 was not transferred. This control demonstrated the

cor-rectness of the sequence of Tn6198.

Discussion

Tn6198 is the first Tn916-like element harbouring a trimethoprim

resistance gene (dfrG) next to the tet(M) gene. The presence of

this transposon in L. monocytogenes is notable because such

transferable elements are rare in this species. Integration of

accessory resistance genes into Tn916 was reported previously

for, among others, Streptococcus pneumoniae,

_{where erm(B)}

confers resistance to erythromycin (Tn6002, originally found in

Streptococcus cristatus and Tn3872) and aphA-3 in addition to

kanamycin (Tn1545

and Tn6003). A combination of the

resist-ance genes tet(M), tet(L) and erm(T) was recently detected on

Tn6079 in the gut of an infant.

_{Apart from antibiotic resistance,}

Tn6009 harbouring the mercury resistance gene mer next to

tet(M) was described in S. aureus

and Tn6087 conferring

resist-ance to both tetracycline and the antiseptic cetrimide bromide

seems to be widespread among Streptococcus oralis strains.

Generally, the presence of more than one antibiotic resistance

gene seems to be an exception in L. monocytogenes, as only a

few cases have been reported, from France, Greece and

Switzer-land. In all these incidents, resistance was plasmid mediated.

Concerning

the

recent

report

of

a

tetracycline-

and

trimethoprim-resistant foodborne L. monocytogenes isolate

from France, the authors did not elaborate whether dfrD is

encoded on plasmid pIP823 or whether there is a connection

to tet(M), which seems to be present on a Tn916-like

trans-poson.

Until now, dfrG was mainly detected in enterococci

and staphylococci,

_{where it was characterized and reported}

in 2005.

As the fragment with dfrG and orfU1 detected in

S. aureus CM.S2 was flanked by inverted and direct repeats, the

authors suggested involvement of an insertion sequence (IS).

Due to a high similarity of the fragment to plasmid pMG1 from

E. faecium, they assumed that it might originate from this

species. However, its equivalent in pMG1 additionally contains

two ORFs in the central region that correspond to orfA and orfB

of IS1485, encoding for transposases.

They seem to have

been lost through a deletion that turned the element into a

mobile

insertion

cassette

where

passenger

genes

with

unknown functions are present instead of transposases.

This

process might be an explanation of how fragment F23-24

ended up in Tn916, turning it into Tn6198. Recently, the same

fragment was also detected in the genome of clinical

Streptococ-cus pyogenes isolates in India.

We demonstrated that both Tn6198 and Tn916 are present in

L. monocytogenes TTH-2007. The fact that nearly identical

Tn916-like elements can coexist in bacterial strains was observed

Table 1. Presence or absence of different types of transconjugants after matings on filters, cheese and salmon from donor L. monocytogenes TTH-2007 to recipients L. monocytogenes 10403S and E. faecalis JH2-2

Recipient Selection Filter mating, L. m. TTH-2007 as donor of: Cheese mating, L. m. TTH-2007 as donor of: Salmon mating, L. m. TTH-2007 as donor of:

Tn6198 Tn916 Tn6198 and Tn916 Tn6198 Tn916 Tn6198 and Tn916 Tn6198 Tn916 Tn6198 and Tn916

L. m. 10403S TMP13 + 2 2 2 2 2 + 2 2 L. m. 10403S TET26 + + + ND ND ND + + + L. m. 10403S TET26+TMP13 + 2 + + 2 + + 2 + E. f. JH2-2 TMP13 2 2 2 2 2 2 2 2 2 E. f. JH2-2 TET26 2 + + 2 2 2 2 2 2 E. f. JH2-2 TET26+TMP13 + 2 + 2 2 2 2 2 2

L. m., L. monocytogenes; E. f., E. faecalis; TMP13, 13 mg/L trimethoprim; TET26, 26 mg/L tetracycline;+, presence of transconjugants; 2, absence of

transconjugants; ND, not determined.

before for clinical E. faecium isolates.

Both transposons were

transferable together or separately by conjugation in vitro. In

contrast, although we detected dfrG fragment F23-24 as a

circu-lar form (Figure S1, available as Supplementary data at JAC

Online), it seems not to be transferable on its own. This is in

ac-cordance with the previous finding that intra-species conjugal

transfer of dfrG from donor strain S. aureus CM.S2 was not

suc-cessful.

_{In our study, dfrG was only transferred together with}

the Tn916 part of Tn6198, which provided the required elements

for conjugation.

In addition to horizontal gene transfer by mating on filters,

transmission of Tn6198 and Tn916 was demonstrated within

the smear of semi-mature cheese stored at 228C and on the

surface of smoked salmon at both 98C and 228C. This is the

first description of such an event on cheese and salmon surfaces.

However, further tests with different storage times and

tempera-tures should be performed to detect the physical parameters

influencing the transferability of Tn6198. The transfer of the

con-jugative transposon Tn6198 on cheese surfaces is an especially

important finding, as Listeria spp. can be in close contact with

high concentrations of enterococci and other bacteria during

cheese ripening and storage. Despite the fact that total bacterial

counts on salmon are generally lower than on cheese, both food

matrices provide favourable conditions for horizontal gene

trans-fer where Listeria can act both as donor and recipient. Even

though it is not known where the clinical strain TTH-2007

received Tn6198, as almost all L. monocytogenes infections in

humans are caused by the consumption of food, it is important

to determine the transferability of mobile genetic elements in

the foodstuffs where Listeria spp. occasionally cause problems.

In conclusion, we detected a clinical multidrug-resistant

L. monocytogenes isolate associated with a novel conjugative

Tn916-like transposon Tn6198. In addition to providing its

com-plete sequence (GenBank accession no. JX120102), we

demon-strated its transferability both in vitro and in situ on the surface

of salmon and cheese, respectively, indicating further

opportun-ities for spread.

Acknowledgements

We are grateful to Professor Jacques Bille from the Centre Hospitalier Universitaire Vaudois (CHUV) in Lausanne for providing L. monocytogenes isolates.

Funding

This work (part of the project ‘BactFlow’) was supported by the Compe-tence Center Environment and Sustainability (CCES) directed by ETH Zurich, 8092 Zurich, Switzerland.

Transparency declarations

None to declare.

Supplementary data

Table S1 and Figure S1 are available as Supplementary data at JAC Online (http://jac.oxfordjournals.org).

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